The ongoing threat of recent avian influenza outbreaks combined with the possibility of recombination with human influenza suggests that the global spread of a novel pandemic is imminent. The two types of human influenza (A and B) have historically-proven potential to cause epidemic disease which can be lessened by antiviral drugs that can effectively target and rapidly inhibit viral propagation. We propose to optimize and adapt in vitro biochemical assays for high-throughput screening additional antiviral compounds effective against four validated, high-priority biochemical targets: 1) neuraminidase, 2) NS1, 3) polymerase cap snatching activity and 4) polymerase endonuclease activity. In addition to the four enzymatic targets, we propose to optimize two whole cell assays with unique, less explored phenotypic endpoints. The first measures cell leakage through current measurements of ion channel voltage and the second measures viral-induced cell death. These novel assays will be validated for the discovery of new inhibitory lead compounds. The four biochemical assays will be complemented with the two cell-based assays to confirm and extend the biochemical results, totaling six diverse assays for translation to HTS. These assays are critical for identifying new lead compounds for influenza antivirals and have been designed with the potential to be effective for a broad spectrum of both influenza A and B types, including newly emergent, highly pathogenic influenza strains. Biochemical assay designs for neuraminidase, NS1, and polymerase functions will exploit the high sensitivity and low background of fluorescence and luminescence-based detection for identification of new inhibitory lead compounds. The assays systems will be employed in BSL-1 and BSL-2 environments using laboratory adapted influenza strains or recent, low pathogenicity human isolates. As assays are adapted, we propose to validate the HTS methods using small screens with potential antiviral drugs. The compounds to be screened initially include a 3,000 compound diversity set tailored for the target from our discovery library of over 100,000 commercial compounds, which were selected for structural diversity and drug-likeness. The focus of this proposal is on the design and optimization of novel, robust assays that can be adapted to an automated liquid HTS system. [unreadable] [unreadable] [unreadable]